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1980 The ccO urrence of Alliinase in Selected Bacterial Strains. Lawrence A. Reily Louisiana State University and Agricultural & Mechanical College
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Recommended Citation Reily, Lawrence A., "The cO currence of Alliinase in Selected Bacterial Strains." (1980). LSU Historical Dissertations and Theses. 3496. https://digitalcommons.lsu.edu/gradschool_disstheses/3496
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University M icrofilm s International
300 N. ZEEB ROAD, ANN ARBOR, Ml 48106 18 BEDFORD ROW, LONDON WC1R 4EJ. ENGLAND 8021758
REILY, LAWRENCE A.
THE OCCURRENCE OF ALLIINASE IN SELECTED BACTERIAL STRAINS
The Louisiana State University and Agricultural and Mechanical Col. PH.D. 1980
University Microfilms International300 N. Zeeb Road, Ann Arbor, MI 48106 18 Bedford Row, London WC1R 4EJ, England THE OCCURRENCE OF ALLIINASE IN
SELECTED BACTERIAL STRAINS
A Dissertation
Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy
in
The Department of Food Science
by Lawrence A. Reily B.S., Louisiana State University, 1971 .S., Southeastern Louisiana University, 1975 May, 1980 ACKNOWLEDGMENTS
The author wishes to express his indebtedness to Dr.
R. M. Grodner, his major professor, and to Dr. H. D.
Braymer, his minor professor, for their contributions to this pursuit. He is grateful to the faculty, staff, and students in the Department of Food Science, whose associa tions have been both enlightening and enjoyable.
ii TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS ...... ii
LIST OF TABLES ...... iv
LIST OF FIGURES ...... v
ABSTRACT ...... *...... vi
INTRODUCTION ...... 1
REVIEW OF LITERATURE ...... 2
MATERIALS AND MET BOD S ooo9ooooeeeeooooa*»*<>oo«B»eoo« 20
Alliinase Occurrence ...... 20
Enzyme Induction ...... 24
Inhibition Tests ...... 25
Alliinase Identity ...... 26
RESULTS AND DISCUSSION ...... 31
Alliinase Occurrence ...... 31
Inhibition Tests ...... 36
Enzyme Induction ...... 38
Alliinase Identity ...... 41
S u m m a r y ...... 46
LITERATURE CITED ...... 47
APPENDIX ...... 53
VITA ...... 57
iii LIST OF TABLES
Table Page
1. Microorganisms Tested for Alliinase Activity ...... 32
2. Quantities of Allicin Produced in ATB Cultures ...... 35
3. Growth of Selected Organisms on S T A ...... 37
4. Alliinase in Cell Crops of Selected Organisms ...... 39
5. Pyruvate Production by Cells of Escherichia coll 1 »»•••■•■•••«••••••••••••••• 44
6. Purification of Alliinase and Cystathionase Activities ...... 45 LIST OF FIGURES
Figure Page
1. Allicin (allyl thiosulfinate)...... 3
2. Alliin [(+)-S-allyl-L-cysteine sulfoxide] .... 5
3. The alliinase reaction ...... 6
4. Cystathionase activity in Neurospora crassa: g-cleavage and y-cleavage ...... 14
v ABSTRACT
Alliinases have been studied in animals and plants,
but relatively little attention has been given to bacte
rial alliinases. Therefore, an investigation was
initiated to determine the prevalence and nature of
alliinase systems in bacteria. A method of determining
alliinase activity in bacteria was developed. The proce dure was based on qualitative or quantitative detection of allicin, by a color reaction with N-ethylmaleimide.
Alliinase was found to be present in some strains and absent in others. In still other strains, alliinase
seemed to be a variable characteristic. It was speculated that alliin cleavage in bacteria might be accomplished via an inducible cystathionase. Both alliinase and cystathio nase activities were found in cell crops of E. coli and B. subtilis, when these organisms were cultivated in a glu- cose-salts medium. Also, alliinase and cystathionase activities were found to be co-purified from E. coli cells, through a sixty-fold purification procedure. These pieces of evidence are in support of the theory that bacterial alliinase might be attributable to a cystathionase system.
vi INTRODUCTION
Garlic (Allium sativum L.) possesses an antimicrobial
principle known as allicin (allyl thiosulfinate). This
substance is produced enzymatically when garlic is cut or
crushed, and is the main constituent of the characteristic garlic odor. Allicin is produced by cleavage from its parent compound, alliin (S-allyl-L-cysteine sulfoxide), via the specific garlic enzyme, alliinase. Alliinase ac tivity has also been demonstrated in other plant species, in animals, and in bacteria.
Relatively little research has been done concerning the occurrence of alliinase systems in bacteria. There fore, an investigation was initiated to determine the prevalence of these enzymes in various bacteria. It was further desirable to determine the usefulness of knowing whether or not an organism possesses alliinase activity.
The presence or absence of an alliinase could prove to be useful in identification schemes. Also, a selective action might be found in alliin-containing media: the inhibitory action of the allicin, which is produced enzymatically, might selectively prevent the growth of organisms which possess alliinases. In an effort to understand the significance of alliinases in bacteria, a study was also made, to determine the nature of these enzyme systems.
1 REVIEW OF LITERATURE
The substance responsible for the antimicrobial
properties of garlic is present in expressed garlic juice,
in aqueous extracts of chopped garlic (1), and in vapors
from crushed garlic (59). Cavallito and Bailey (9) ex
tracted the active principle from garlic and called the
compound "allicin". They described the substance as an unstable oil, having the characteristic odor of garlic and
exhibiting antibacterial properties. Cavallito et al.
(10) obtained allicin from fresh garlic in a yield of 0.3 to 0.4%. Cavallito et al. (11) studied the chemistry of allicin in an effort to elucidate the compound's struc ture. They proposed that allicin might be the allyl ester of allyl thiosulfenic acid (Fig. 1). The structure was subsequently confirmed by Small et al. (48), who synthe sized allicin and several other alkyl thiosulfinates and demonstrated the antimicrobial activities of these com pounds. The antibacterial nature of allicin has been the object of much study. Allicin is active against a wide spectrum of organisms, and probably operates by binding sulfhydryl groups, which occur at the active sites of certain essential enzymes (38). Inhibition of bacterial growth by allicin has been demonstrated in dilutions as low as 8.0 yg/ml (9). The effect of allicin tends to be bacterial stasis (11), but, in sufficient concentration,
2 3
c h 2=c h -c h 2-s -s -c h 2-c h =c h 2
Fig. 1. Allicin (allyl thiosulfinate) 4 the compound can be lethal to microorganisms, killing even bacterial spores (59).
Intact garlic has relatively little odor, but when garlic tissue is cut or crushed, the characteristic odor of allicin develops. This observation implies that allicin is produced or released when garlic is damaged.
Rundqvist (41) demonstrated that allicin is enzymatically cleaved from a parent compound in garlic. He called this precursor "alliin" and mistakenly identified it as a glucoside. Stoll and Seebeck (51) subsequently isolated the parent compound in crystalline form and adopted the epithet "alliin" for the substance, as proposed by
Rundqvist. Uchida et al^. (56) reported extraction of alliin from garlic in 0.5 to 0.8% yield. Stoll and
Seebeck (51) studied the in vitro synthesis of alliin and its isomers and produced (+)-S-allyl-L-cysteine sulfoxide
(Fig. 2), which is identical to natural alliin.
A mechanism for the enzymic cleavage of alliin was proposed by Stoll and Seebeck (54) (Fig. 3). The alliinase of garlic is thought to cleave allyl sulfenic acid from alliin as the first step of the reaction. Two molecules of this unstable intermediate then undergo a nonenzymatic association, with the elimination of one molecule of water, to form the thioester, allicin. The other intermediate, a-aminoacrylic acid, is also unstable and reacts with water to liberate ammonia and thus form pyruvic acid. Spare and Virtanen (50) questioned the 5
O II CH2=CH-CH2-S-CH2“CH-COOH nh2
Fig. 2. Alliin [ (+)-S-allyl-L-cysteine sulfoxide]. Allylsulfenic Allicin acid
II CH I CH, I z Alliinase s=o > + I CH- l 2 H,N-CH 2 I COOH
Alliin 2 N H 3 + 2 H 20 2 H2N-C > + COOH CH, a-Aminoacrylic l 3 2 C=0 acid l COOH
Pyruvic acid
Fig. 3. The alliinase reaction (54). 7 proposed reaction scheme. Their mass spectroscopy studies failed to detect the intermediate thiosulfenic acid in alliinase reaction mixtures. Therefore, if this compound is produced it must be extremely short lived. Spare and
Viroanen indicated that the alliinase reaction might occur by a mechanism different from that proposed previously, bxit conceded that their spectral data failed to suggest an alternate mechanism. Stoll and Seebeck (52, 53) re ported some of the characteristics of garlic alliinase.
The enzyme may be extracted into water from chopped garlic and purified by precipitation at its isoelectric point, pH 4.0. The garlic enzyme is inactivated by organic sol vents such as ethanol or diethyl ether. Alliinase is most st. ble at pH 6.4, but preparations stored at this pH and c -.emperature of 3°C lose their activity within 14 days.
On y 50% of the activity is retained at this pH after 30 ca's of storage at -10°C. Mazelis and Crews (30) showed i t xt alliinase activity could be protected by addition of
10% v/v glycerol to the enzyme preparation.
The alliinase reaction is quite rapid, degrading more than 80% of the substrate in 2 min in a 1.0% alliin solu tion. Garlic alliinase is active in the pH range of 5.0 tc- 8.0, and changes within this range cause little differ- er :e in activity. The actual pH optimum has been reported j.s pH 6.5 (30). The optimum temperature for the alliinase
e action is 37°C, but the enzyme remains active from 0 to
>0°C. It has been demonstrated that garlic alliinase has 8
requirements for pyridoxal phosphate (23) and Mg++ (30).
The specificity of garlic alliinase has been summa
rized by Stoll and Seebeck (35), who listed the following
requirements for substrates:
(1) They must be derivatives of a-amino-8-mercapto- propionic acid (cysteine). Substances which contain a homolog of cysteine such as homocysteine or penicillamine are not split by alliinase. (2) The sulfur atom of the cysteine derivatives must be linked to an aliphatic group such as ethyl, propyl, etc. Derivatives having an aromatic group in this position are not split. (3) The amino group of the cysteine portion of the molecule must not be sub stituted. N-aniloformylalliin is not attacked by alliinase. (4) The sulfur atom of the cysteine derivative must be present in the form of a sulfox ide. Desoxyalliin resists enzymic cleavage, whereas the double bond in the allyl group does not appear to be essential for cleavage to take place.
Based on further experimentation, Stoll and Seebeck
(54) also noted the following:
(1) The compound attacked must be derived from natural, levorotatory (-)-L-cysteine. Derivatives of dextrorotatory (+)-D-cysteine cannot be broken down by means of alliinase. (2) The sulfur atom may be present either in the optically active or in the racemic form, but only compounds with a configu ration corresponding to natural alliin undergo rapid degradation.
The occurrence of S-substituted alkyl cysteine sulf oxides has been reported in the free amino acid pools of
several plant species. Alliin, in garlic, was the first compound of this group to be discovered (77) . Further
studies have revealed alliin-like compounds in other members of the Liliaceae and in species of the Umbellif- erae, Mimosae, and Cruciferae, as well. 9
Following demonstration of the antibacterial proper
ties of garlic, reports appeared of antibacterial activity
in related plant species. Lovell (26) noted that vapors
from crushed onion had bactericidal effects similar to,
but not as potent as, garlic. Cavallito, Bailey, and Buck
(10) found that neither allicin nor its precursor, alliin,
occur in onion. Vilkki (51) demonstrated the presence of
an alliin-like substance in onions and noted that this
compound yields pyruvic acid, ammonia, and the odor of
onion when incubated with a drop of fresh onion juice.
Fujiwara et al_. (20) analyzed the thiosulfinates
(R-SO-S-R') from several Allium species. The alkyl
(R and R 1) groups reported by these workers included CH3-,
CH3-CH=CH-, and CH3-CH2-CH2-, but no CH3-CH2-group was
detected. Renis and Henze (62) studied cysteine deriva
tives in yellow onion bulbs. Alliin (S-allyl-L-cysteine
sulfoxide) was not detected, but desoxyalliin (S-allyl-L-
cysteine) and dihydroalliin (S-propyl-L-cysteine sulfoxide) were present. Structures of alliin-like compounds dis
cussed in the text are presented in the Appendix, Table 1A.
Virtanen and Matikkala (58) isolated S- methylcysteine
sulfoxide and S-n-propylcysteine sulfoxide from onion
(Allium cepa). They also found S-propyl-enyl-cysteine
sulfoxide in onion, and showed that this compound is the precursor of the lachrymatory factor. Carson and Wong (6)
isolated and identified (+)-S-methyl-L-cysteine sulfoxide and (+)-S-n-propyl-L-cysteine sulfoxide from onion, 10
confirming previous reports and establishing the conforma
tions as the dextrorotatory isomers for both of these compounds.
Morris and Thompson (33) were first to report the occurrence of (+)-S-methyl-L-cysteine sulfoxide in the roots of turnips (Brassica rapa), and isolated 5 g of the substance from 100 pounds of turnip roots. It was subse quently shown that (+)-S-methyl-L-cysteine sulfoxide exists in other members of the Cruciferae, particularly in other species of the Brassica genus (12, 28, 29). The compound war not detected in protein hydrolysates prepared from these plants and, therefore, probably occurs only in the non-prolem fraction (34). The methyl analog of alliin has also been found in other plant families, such as
Compositae in lettuce, Lactuca sativa L.), Umbelliferae
(in honewort, Cryptotaenia japonica Hasskare) (26), and
Leguminosae (In bean, Phaseolus vulgaris L.) (55). Allyl- and propylcysceine sulfoxides have been reported only in
Liliaceae (r 6 . The only known occurrence of ethyl- cysteine suj f ucide is in Ipheon uniform™, another member of the Liliaceae (60).
Several workers have studied alliinase, the S-alkyl-
L-cysteine sulfoxide lyase of garlic (E. C. 4.4.1.4).
Stoll and Seebeck (52) were the first to report isolation of this enzyme, and they noted some of its more obvious characteristics, such as temperature and pH relationships.
Goryachenkova (22) demonstrated a requirement for pyridoxal 11
phosphate in the alliinase reaction. Mazelis and Crews
(30) studied alliinase preparations which were more highly
purified than those previously employed. These authors
listed several characteristics of the garlic enzyme. They
confirmed the pyridoxal phosphate requirement and noted
that this cofactor is tightly bound to the protein portion
of the enzyme and remains attached unless stringent puri
fication procedures are used. Additionally, Mazelis and
Crews confirmed the observations that activity of the
enzyme is stimulated by magnesium ions and is greatest at
pH 6.5. Alliinase will cleave several alkyl cysteine
sulfoxides, but Vmax is greatest with its natural sub
strate, (+)-S-allyl-L-cysteine sulfoxide.
Some plant species, other than garlic, possess enzyme
systems which can produce allicin from its parent compound.
Some other members of the Liliaceae exhibit such enzyme
activity. Onions, for example, display a phenomenon
similar to garlic; odor and lachrymatory properties do not develop until the bulb is damaged. This fact implies that
a cleavage enzyme is also involved in onions. Virtanen
and Matikkala (58) confirmed this hypothesis by preparing
an enzymatically active precipitate from aqueous onion
extract. Schwimmer et aJ. (44) issued the first report of onion enzyme purification. The cleavage enzyme from onion
is similar in specificity to the garlic enzyme, but
differs in some of its other characteristics. On Sephadex
fractionation, the onion enzyme gives two peaks of 12
activity (58). Spare and Virtanen (50) accept this as
evidence that two distinct enzymes with different molecu
lar weights may exist, citing also that, at pH 4.5, there
is activity in both the precipitate which forms and in the
supernatant. These workers concede, however, that these phenomena could also result from disintegration of the enzyme.
Several plant species not in the Liliaceae also possess enzymes which cleave alkyl cysteine sulfoxides. A woody ornamental shrub, Albizzia lophanta, which belongs to the Leguminosae has an enzyme in its seeds with alliinase activity (21). The Albizzia enzyme differs from garlic alliinase in its pH optimum of pH 8.5 and its broader specificity, but, similarly, has a requirement for pyridoxal phosphate. This enzyme will cleave L-cysteine and various, related compounds, including S-substituted alkyl cysteines and their sulfoxides, cystathionine, and djenkolic acid (45). Cysteine sulfoxide lyases have been discovered in several members of the family Cruciferae, particularly in some Brassica species. These include broccoli (B. oleraceae var. pompejana), turnip (B. rapa), cauliflower (B. oleraceae var. botritis), brussels sprouts
(B. oleraceae var. germifera), cabbage (B. oleraceae var. capitata), and rutabaga (B. napobrassica) (28, 29). The pH optima for these Cruciferae enzymes are in the alkaline range, as is the case with the enzyme in Albizzia. Alkyl cysteine sulfoxides, as mentioned earlier, also are found 13 in some of these Brassica species, coexisting with the cleavage enzymes. The in vivo degradation of these poten tial substrates is probably prevented by the high pH requirements of the enzymes. Mazelis (28) showed that activities of the alkyl cysteine sulfoxide lyases of
Brassica species are almost non-existent at a pH near neutrality.
Another place in the plant kingdom where an alkyl cysteine sulfoxide lyase is found is in the shiitake mush room (Lentinus edodes) (24) . This enzyme has a high pH optimum like the Albizzia and Brassica enzymes and, like wise, requires pyridoxal phosphate. A few other fungi also possess lyases which cleave alliin or other analogs of cysteine. Penicillium corymbiferum is the etiological agent of bulb decay of garlic. Durbin and Uchytil (14) reported a cysteine sulfoxide lyase in this fungus. The enzyme has properties similar to garlic alliinase, includ ing specificity, pH optimum (pH 6.5), and Michaelis con stant. The K values with alliin as substrate are 6 mM m for garlic enzyme and 5.8 mM for Penicillium corymbiferum enzyme. The fungal enzyme also requires pyridoxal phos phate as a cofactor. Another fungus, Neurospora crassa, has two distinct lyases which split an analog of cysteine, cystathionine (18) (Fig. 4). One of these is a y-cleavage enzyme which produces cysteine and a-ketobutyrate from cystathionine. The y-cleavage enzyme is thought to be reversible and act also as a condensing enzyme to 14
COOH i H 2N-CH l CH, I z COOH c h 2 I SH h 2n -c h Homocysteine 4 h 2 6-cleavage
?H 2 H,N-CH 2 l o=c COOH I COOH Cystathionine Pyruvate
COOH i o=c I CH, COOH I *■ i CH, H-N-CH I a-Ketobutyrate CH, I CH, Y-cleavage l S l CH, l SH H,N-CH I l CH. COOH I H,N-CH Cystathionine 2 l COOH Cysteine
Fig. 4. Cystathionase activity in Neurospora crassa: 3-cleavage and ycleavage (16). 15 synthesize cystathionine (18). The other enzyme catalyzes a 8-elimination reaction which yields homocysteine and pyruvate from cystathionine and is analogous to the 8- cleavage of alliin by alliinase. The 8-cleavage and y- cleavage enzymes of Neurospora are probably involved in the methionine pathway. The ability of Neurospora to grow without exogenous methionine has been attributed to its capacity to carry out a reciprocal pair of reactions in which cystathionine is formed from cysteine and homoserine by reversal of the y-cleavage enzyme, then cleaved by 8- elimination to homocysteine, pyruvate, and ammonia (18).
It has also been speculated that Neurospora may utilize sulfur by transferring S-methyl- groups from S-methyl- cysteine, a metabolite which occurs naturally in this organism (37).
The 8-cystathionase of Neurospora cleaves several compounds in addition to cystathionine, including cystine, homocystine, thioethers, and hydroxyamino acids (17).
Several fungi, in addition to those already mentioned, are known to possess enzyme systems which cleave cysteine derivatives (62). Seopularcopsis brevicaulis, Aspergillus niger, and Penicillium notatum act on a number of thio ethers and convert them to thiols. These reactions in clude the conversion of S-methyl-, S-ethyl-, and S- propylcysteine to methane-, ethane-, and propanethiol, respectively. 16
Alliinase activity has been demonstrated in the animal
kingdom as well as in the plant kingdom. Specifically,
alliin-cleaving enzymes have been found in mammalian
systems. Rothlin (40) showed that the odor of garlic develops in the breath of laboratory animals which have received intravenous alliin injections. The enzyme which
is responsible for alliin cleavage in mammals is probably the cystathionase that is found in the liver. This enzyme has been shown to cleave cystine, lanthionine (7), cysteine, djenkolic acid, allocystathionine, cysteine methyl ester, and several S-alky1-cysteines (4). Cysta thionase may also be responsible for the degradation of
S-(1,2-dichlorovinyl)-L-cysteine, which is catalized by liver enzymes (2). Reports of enzymes which cleave cysteine derivatives have, by no means, been restricted to eucharyotic organisms. Enzymes of this type have been noted in bacteria, as well as in higher organisms. Von
Euler (16) found that the odor of garlic (allicin) devel oped in a culture of Escherichia coli when 0.2% alliin was added. Murakami (35) reported the ability of Bacillus subtilis to cleave S-allyl- and S-methylcysteine sulfox ides. Nomura et al_. (36) found that a Pseudomonas cruciviae strain, grown in a medium with S-methylcysteine sulfoxide as the major carbon and nitrogen source, produces an enzyme which cleaves pyruvate from S-methyl-,
S-ethyl-, and S-allylrysteine, and the corresponding sulfoxides. Cells grown on any one of these substrates 17 were also adapted to the others, as evidenced by absence of a lag period. Other compounds utilized by the pseudo- monad were S-n-propyl-, S-n-butyl-, S-isobutyl-, S-n-amyl-, and S-isoamyl-L-cysteine, and isovalthine. It has been reported that several bacteria, including Escherichia coli,
Proteus vulgaris, Pseudomonas aeruginosa, Clostridium perfringens, and Clostridium tetani possess enzymes which cleave methanethiol and pyruvate from certain thioether derivatives, such as methionine, S-methylcysteine, a-oxy- y-methiobutyric acid, and y-methiobutyric acid (31, 32).
Yamada and Kumagai (61) patented a process by which S- allyl-L-cysteine could be produced enzymatically from pyruvate, ammonia, and allylthiol. The process employs reversal of microbial (Enterobacter aerogenes) cysteine desulfhydrase activity. However, the corresponding cleavage reaction is presumably not accomplished by cysteine desulfhydrase in bacteria. The bacterial enzymes studied by Mitsuhashi (31) and Nomura et al. (36) proved unable to cleave cysteine. Cavallini et al. (8) demon strated that the cysteine desulfhydrase activity in liver is effected by cystathionase. Based on the work of Flavin
(17), these authors extended their observation to the desulfhydrase system in Escherichia coli. The cystathio nase systems in bacteria differ from those found in fungi.
Delavier et al. (13) noted that Neurospora and a yeast had both 6-cystathionase and y-cystathionase. An Escherichia coli and a Salmonella strain had only the 8-cleavage 18
enzymes. Mutant strains of these bacteria were derived
which did not possess a cystathionase.
Several workers tested bacterial action on S-(l,2-
dichlorovinyl)-L-cysteine (DCVC). Escherichia coli B was
found to contain an enzyme which cleaves L-DCVC and its
sulfoxide, DL-(+)-allocystathionine, and L-serine to form
a-keto acids (42, 43). The enzyme has little activity
toward S-ethyl-L-cysteine, D-DCVC, N-acetyl-L-DCVC, and cysteine.
Pyridoxal phosphate occurs in the tissues of plants and animals and in bacterial cells as enzyme-bound or other protein-bound forms. The enzymes which cleave alliin belong to a large group of pyridoxal phosphate enzymes.
The various alliinases can be further characterized by
subgrouping them with the a,8-elimination enzymes. Other examples of enzymes which catalyze a ,8-elimination reac tions are L- and D-serine dehydrases, L- and D-threonine dehydrases, cysteine desulfhydrase, and tryptophanase (49).
Pyridoxal or pyridoxal phosphate is the active center of each of the pyridoxal-dependent enzymes. Groupings in the apoenzyme serve to enhance and lend specificity to the catalytic properties of the coenzyme (49).
Snell (49) has postulated the mechanism of a,8“ elimination reactions. Following formation of the enzyme- substrate complex, electron withdrawal by the pyridoxal phosphate group weakens bonds around the a-carbon. The metal ion which is usually required in these reactions may 19 provide additional electron attraction. This weakening of bonds constitutes activation of the substrate by the enzyme. An electron-attracting group on the 8-carbon will assimilate some of the charge of an extra electron pair.
This allows an anion to be liberated as an intermediate, in equilibrium with pyridoxal, the metal ion, and an a- aminoacrylic acid. The a-aminoacrylic acid then decomposes spontaneously to form an a-keto acid and ammonia. Happold
(23) studied the cleavage of tryptophan by a pyridoxal phosphate enzyme from Escherichia coli. In contrast to
Snell's mechanism, Happold (50) described the intermediate which was formed, as a free radical which propagates it self. Considering the extreme reactivity of free radicals,
Happold's mechanism might explain why intermediate species have not been detectable in alliin-alliinase reaction mixtures. The net effect of the (3-cleavage is that an a-amino acid, with an electronegative substituent on the
8-carbon is converted to an a-keto acid by elimination of the electronegative group and ammonia (2). MATERIALS AND METHODS
Alliinase Occurrence
The detection and quantitation of alliinase activity has been accomplished by several means. Determination of volatile sulfur compounds, pyruvic acid, ammonia, and thiosulfinates have all been used as indices of alliinase activity (19). It was desirable, for this study, to develop a qualitative or semi-quantitative method of de tecting alliinase activity which would be both rapid and convenient. It was felt that detection of one of the products of the alliinase reaction would be the proper avenue to pursue, since detection of a small residual of substrate in the complex mixture of a bacterial culture would be quite difficult. Measurement of pH change, ammonia production, or pyruvate production in such a system was deemed equally formidable. Therefore, a test for the remaining product, allicin, was sought, and was developed in this study.
Rao et al^. (38) reported that allicin could be quan titatively extracted from aqueous solution by chloroform.
Solvent extraction was used by Schwimmer and Mazelis (46) in their study of thiosulfinate production in onions. It was therefore anticipated that extraction could offer a means of removing and concentrating allicin from bacterial cultures being tested. Bensch et al. (3) discovered that
20 21
thiols and thioesters could be detected by reaction with
N-ethylmaleimide (NEM). When the products of this reac
tion are made alkaline, a red color appears. Bensch et al.
used the NEM reaction to develop paper chromatograms and
listed a number of compounds which could be detected by
the method. Carson and Wong (5) developed a colorimetric
assay for thiosulfinates, based on the NEM reaction, and
found that the method could detect 5 X 10"2 umole/ml of
thiosulfinate in isopropanol solution. Schwimmer and
Mazelis (46) used this NEM procedure in their previously mentioned study of onion thiosulfinates. Laboratory tests
showed that allicin, prepared as an aqueous extract of
fresh garlic, could be recovered from nutrient broth by
extraction into chloroform and detected by the NEM reac tion of Carson and Wong (5). Chloroform layered below the aqueous phase due to its greater density, and required removal to another vessel for addition of the test reagents. An extractant less dense than water was desir able, so that the entire test could be carried out in one culture tube. Petroleum ether was found to be a suitable substitute for the chloroform, extracting the allicin and layering above the aqueous phase, allowing direct addition of the test reagents. The actual performance of the test was similar to that used by Carson and Wong, in that the constitution and quantities of reagents were the same.
A suitable medium for testing allicin production by bacterial strains was required. Previous workers (31, 16) 22
had used substrate-salts media, but it was feared that all
of the strains used in this investigation might not grow
in a minimal medium. Therefore, nutrient broth (Difco)
with added alliin was tested and proved acceptable.
A racemic alliin preparation was obtained by synthe
sis, based on the method of Stoll and Seebeck (51). S-
allyl-L-cysteine (ICN or Vega) was oxidized by an excess
of 30% H202. Five milliliters of 30% H202 were used per gram of S-allyl-L-cysteine, and the mixture was allowed
to stand at room temperature for 48 h. The mixture was placed on a rotary evaporator (Buechi) and the liquid evaporated to a syrup in vacuo at 45°C. The alliin thus produced was collected by precipitation with acetone.
Additional alliin could be obtained from the supernatant by repeating the evaporation and precipitation steps. The
synthetic product was washed with acetone, dried in vacuo, and stored in a closed container.
Because alliin is not readily available through commercial channels, the test medium was designed to use the minimum practicable quantity of alliin. The alliinase test broth (ATB) was made by preparing nutrient broth and adding alliin to the solution to a concentration of
2 mg/ml. The medium was not stable to autoclaving and was, therefore, sterilized by passage through a 0.45 ym membrane filter. One milliliter aliquots of the sterile broth were dispensed into sterile 13 mm X 10 mm screw capped test tubes. 23
The Bacillus cultures which were tested were isolated from local soils. The remainder of the bacterial strains were random isolates obtained during plate counts of various seafoods. All of the isolates were tentatively identified according to Bergey's Manual, eighth edition.
The NEM test for the occurrence of alliinase in a bacterial strain was carried out as follows:
1. A sterile 1.0 ml aliquot of ATB was inoculated
from a 24 h nutrient agar slant of the test
organism.
2. The culture was incubated 48 h at 35°C.
3. One milliliter of petroleum ether was added to
the culture, the tube was inverted several times,
and the phases were allowed to separate.
4. 0.5 ml of 0.05 M N-ethylmaleimide (Eastman) in
isopropanol was added to the tube.
5. 0.5 ml of 0.25 M KOH in isopropanol was added.
The color reaction which developed in the ether phase was then rated as positive or negative. Results were read immediately after addition of KOH, as the colored product is unstable. Occasional cultures yielded very faint reactions, which were read as negatives. Only reactions which produced an easily perceptible pink or red color were designated as positive.
For a quantitative NEM test, the color reaction which developed in the ether phase was measured in situ at
515 nm, with a Bausch and Lomb Spectronic 20. Allicin 24 concentration was read from a standard curve, a plot of concentration as a function of absorbance. Data for the standard curve were generated by performing the quantita tive NEM test on allicin standards, prepared in nutrient broth. Allicin was extracted from garlic by the method of Rao et al. (38).
For degradation of residual substrate, alliinase was extracted from garlic by the method of Stoll and Seebeck
(52). Two drops of the alliinase preparation per tube were added to a set of 48 h ATB cultures which duplicated the set used for the quantitation of allicin produced by the cultures. After incubation at 35°C for 30 min, the allicin concentrations were determined by the quantitative
NEM test.
Enzyme Induction
Von Euler (15) was the first to allude to alliinase in bacteria. He described the development of garlic odor when 0.2% alliin was added to an Escherichia coli culture in a synthetic medium. Other workers (31, 36, 42) experi mented with bacterial cells from both complex and minimal media. Their results imply a connection between alliinase activity and growth in minimal media. To test whether alliinase is induced by growth in minimal media, alliinase activity in several bacterial strains was compared between cells grown in complex and minimal media. Nutrient broth
(Difco) (NB) served as the complex medium, and a glucose- salts formulation (GS) served as the minimal medium. The 25
glucose-salts medium was a modification of the substrate-
salts medium of Murakami (35), and consisted of the follow
ing: 3 g K H 2PC\, 6 g N a 2HPC\, 5 g NaCl, 2 g NH..C1, 0.1 g
MgSO^, 8 g glucose, 1000 ml distilled water.
Alliinase induction was determined by testing for
allicin production in cells grown in the complex medium
and in the minimal medium. The cultures to be tested were
inoculated into NB and GS and incubated 24 h at 35°C. The
bacterial cells were collected by centrifugation. Five milliliter aliquots of the GS and NB cultures were centri
fuged and the cells were resuspended in 1.0 ml ATB. The mixtures were incubated 60 min at 35°C. Allicin produc
tion was determined by the NEM test, performed in a manner
identical to that used for determination of allicin in
ATB-grown cultures.
Inhibition Tests
The powerful antimicrobial property of allicin might
lend a selective action to media containing alliin. It was anticipated that organisms which possess an alliinase might produce allicin from alliin, in a concentration which would inhibit their own growth. Other organisms, which do not cleave the antimicrobial agent from its parent com pound, would not be inhibited. This proposed selectivity phenomenon was examined by preparing plates of an agar medium containing alliin, and streaking these plates with various test organisms. The basal medium for selectivity
test agar (STA) was prepared by dissolving 2.3 g of 26 dehydrated nutrient agar medium (Difco) in 90 ml of dis tilled water. The basal medium was sterilized by auto- claving and cooled to 50°C. Ten milliliters of filter- sterilized 5% alliin solution were added to the basal medium, and the completed STA was dispensed into petri dishes. Test organisms were grown for 24 h on nutrient agar slants and streaked onto STA. The inoculated STA plates were incubated at 35°C, and the presence or absence of growth was noted after 24 and 48 h.
Alliinase Identity
There is evidence to imply that the alliinase reac tion, effected by some bacteria, may be carried out by a cystathionase enzyme system. This hypothesis was tested by determining whether cell crops of test organisms, induced to alliin degradation, were also adapted to cysta thionase activity. Cell crops of Escherichia coli 1 were grown in 50 ml each of GS and NB for 48 h at 35°C. Meth odology from the enzyme induction study was used to confirm that alliinase was present in the GS-grown cells, but not in the NB-grown cells. Cells from each of the cultures were then assayed for cystationase activity. The cystathionase reaction was monitored by detecting pyruvate production. Cleavage of alliin was similarly tested, as a reference. Cells harvested from 5 ml aliquots of culture fluid were suspended in 1.0 ml portions of (+)-L-cysta- thionine (United States Biochemical) solution or alliin solution (5 rag/ml of the substrate, in distilled water). 27
Reagent blanks consisted of cells suspended in distilled water. These preparations were incubated 60 min at 35°C.
Occurrence of pyruvate in these reaction mixtures was con firmed by ascending paper chromatography, by the method of
Nomura et al. (36). The reaction was stopped by addition of 0.2 ml cold 10% HC10,,. Any precipitate was removed by centrifugation. The solution was neutralized with 4 N
KOH, centrifuged, and the precipitate was removed. One milliliter of saturated 2,4-dinitrophenylhydrazine in 2 N
HC1 was added. The hydrazone was extracted into 5 ml of ethyl acetate. The acetate layer was extracted with 1 ml of 0.2 M Na2C03. The aqueous layer was acidified with 4 N
HC1, and the hydrazone was extracted into 0.4 ml of ethyl acetate. A sample of pyruvic acid (Baker) (2 mg/ml, in distilled water) was prepared similarly, to serve as a standard. The extracts were spotted on Whatman No. 1 chromatographic paper, and the chromatograms were devel oped as described by Nomura et al. (36), with n-butanol- ethanol-0.1 N NaHC03 (10:3:10).
Quantities of pyruvate produced by the harvested cells were determined spectrophotometrically. The method of Schwimmer and Weston (47) was the basis of the quantita tive pyruvate test. One milliliter of 0.125% 2,4-dinitro phenylhydrazine (Eastman) in 2 N HCl was added to the 1 ml test mixture. One milliliter of distilled water was then added, and the preparation was incubated for 10 min at
35°C. Five milliliters of 0.6 N NaOH was added. The 28 absorbance of the sample was measured at 420 nm on a Spec- tronic 20 (Bausch and Lomb). Pyruvate concentration was read from a standard curve which had been plotted with data derived from reagent blanks to which varying amounts of pyruvic acid (Baker) had been added. Cells of Bacillus subtilis 1 and Pseudomonas aeruginosa 1 were grown in GS and tested, qualitatively, for pyruvate production from cystathionine. The test procedure was identical to that used for Escherichia coli, except that spectrophotometric measurement was not employed. The test was evaluated visually, comparing the test preparation to an unincubated control. A positive test was indicated by development of the reddish-brown color of the phenylhydrazone. A light straw color characterized the negative test.
In order to test whether alliinase and cystathionase activities might be accomplished by the same enzyme, the enzyme was extracted from GS-grown cells of Escherichia coli 1 and carried through a purification procedure. At various stages of the procedure, the ratios of specific activities (cystathionase/alliinase) were determined.
Enzyme purification was based on the method of Nomura et al. (36) . Cells were grown in still culture, from 10% inocula, in GS. Incubation was 48 h at 35°C. The cells were harvested by centrifugation, washed three times with
100 ml of 0.9% NaCl solution, and acetone-dried. The acetone-dried cells (0.8 g) were extracted with 4 ml of distilled water for 20 min. All manipulations in the 29 purification scheme were conducted at or near 0°C. The mixture was centrifuged at 12,000 X g for 15 min. The supernate was then diluted with 10 ml of distilled water.
To the crude extract, 3.3 ml of 0.4% protamine sulfate
(United States Biochemical), pH 7.0, were added. The precipitate was collected by centrifugation and washed twice with 4.7 ml of distilled water. The enzyme was eluted into 5 ml of 0.1 M K 2HP(\, and the mixture was centrifuged for 10 min at 12,000 X g. Ammonium sulfate
(1.1 g) was added to the eluate, the mixture was centri fuged and the precipitate was discarded. This was followed by addition of 0.61 g more ammonium sulfate. The precipi tate was collected by centrifugation and dissolved in
2.5 ml of 0.05 M Tris buffer, pH 8.8. Excess ammonium sulfate was removed by passage through a column of Sephadex
G-50 (Pharmacia). The enzyme was then adsorbed with 1.5 ml of calcium phosphate gel (20 mg/ml, dry weight). The gel was collected by centrifugation and washed with 2.5 ml of distilled water. The enzyme was eluted with 2 ml of
0.1 M K 2HP(\ and 2 ml of 0.25 M K2HP0lf. The eluates were combined. To this solution, 1.1 g of ammonium sulfate were added and the precipitate was removed by centrifuga tion. An additional 0.22 g of ammonium sulfate were then added and the precipitate was dissolved in 3 ml of 0.05 M
Tris buffer, pH 8.8, and passed through a Sephadex G-50
(Pharmacia) column. 30
Enzyme activities were detected as pyruvate produc tion. Reaction mixtures for the assays contained 100 pmoles of Tris buffer, pH 8.8, 2.0 pmoles of (+)-L-cysta- thionine or 4.0 pmoles of racemic alliin, and 0.1 ml of the enzyme preparation, in a total quantity of 1.0 ml.
The test mixtures were routinely incubated for 10 min at
35°C. Under these conditions, degradation of substrate was found to be essentially linear with time, for at least
15 min. Pyruvate was assayed spectrophotometrically, by the method described earlier for the intact-cell studies.
Determinations of protein concentration were based on the procedure of Lowry et al_. (27), using a standard of bovine serum albumin (Sigma). Specific activities, under the reaction conditions listed, were calculated as micromoles of pyruvate produced per minute per microgram of protein. RESULTS AND DISCUSSION
Alliinase Occurrence
Bacterial isolates were grown in alliin-containing media (ATB), and the culture fluids were tested for NEM- reactive substances. Uninoculated controls and control cultures grown in NB gave negative NEM tests. The experi ment showed that there is variability among bacteria, in their alliinase production. Concomitant with NEM tests, positive cultures had the characteristic odor of garlic.
Negative cultures lacked this odor. Therefore, the com pound produced by degradation of alliin, and responsible for the positive NEM tests, was surmised to be allicin.
The production of allicin in garlic occurs via a 8-cleavage from alliin, and bacteria presumably degrade alliin by a similar mechanism.
A number of the test isolates were identified, in an effort to correlate the presence or absence of alliinase systems in bacteria with phylogenetic groupings. The identified isolates and their alliinase capabilities are listed in Table 1. Several of the organisms gave incon sistent results in the test for alliinase. The test was sometimes positive and sometimes negative for these strains. Their occurrence is also reported in Table 1.
This phenomenon could not be attributed to aging of the cultures, nor could contaminating microorganisms be
31 32
Table 1
Microorganisms Tested for Alliinase Activity
Organism Alliinase
Pseudomonas aeruginosa 1
Pseudomonas aeruginosa 2
Flavobacterium rigense
Serratia marcescens +
Enterobacter cloacae +
Escherichia coli 1 +
Escherichia coli 2 +
Escherichia coli 3 +
Escherichia coli 4 +
Bacillus subtilis +
Bacillus megaterium 1 -
Bacillus megaterium 2 -
V- Bacillus circulans 1 -
Bacillus circulans 2 -
Bacillus cereus +
Bacillus firmus +
Bacillus sphaericus -
Bacillus brevis -
Staphylococcus aureus 1
Staphylococcus aureus 2
Staphylococcus epidermidis NC
Staphylococcus sp. 1 + 33
Table 1 (continued)
Organism Alliinase
Staphylococcus sp. 2 +
Staphylococcus sp. 3 +
Staphylococcus sp. 4 NC
Staphylococcus sp. 5 -
Staphylococcus sp. 6 -
Staphylococcus sp. 7 -
Staphylococcus sp. 8 -
Staphylococcus sp.9 NC
Staphylococcus sp. 10 0
Staphylococcus sp. 11 -
Staphylococcus sp. 12 -
Staphylococcus sp. 13 -
Staphylococcus sp. 14 +
Micrococcus luteus
Symbols: + , positive? negative? 0, no growth? NC, not consistent in multiple tests. 34 demonstrated by microscopic or cultural means.
No apparent relationship exists between alliinase activity and identity of the test organisms. Neither is any parallel evident between alliinase and the tests used in identification of the isolates. However, the testing of a large number of cultures, coupled with statistical analysis of the accumulated data could, conceivably, demonstrate a usefulness for alliinase testing in identi fication of bacteria.
In the quantitative study of allicin production by several organisms, allicin concentrations in ATB cultures ranged from 0.01 to 0.07 mg/ml (Table 2). The lower limit of allicin concentration which could be detected visually, by the NEM test (a red color in the petroleum ether layer), was found to be roughly 0.02 mg/ml. The results of the qualitative survey may, therefore, be interpreted in terms of this limiting value. Positive cultures represent allicin concentrations of 0.02 mg/ml or greater; negatives indicate less than 0.02 mg/ml of allicin in the culture fluid.
The theoretical yield of allicin in an ATB culture is
0.44 mg/ml. The cultures assayed by the quantitative NEM test contained only 2 to 16% of the theoretical yield.
This prompted investigations to determine the reason for such low yields. It was found that addition of garlic alliinase increased the allicin concentration in the test cultures to the level predicted by the theoretical yield. 35
Table 2
Quantities of Allicin Produced in ATB Cultures
Organism Allicin (mg/ml)
Escherichia coli 1 0.045
Bacillus sphaericus 0.010
Bacillus subtilis 0.020
Micrococcus luteus 0.014
Pseudomonas aeruginosa 2 0.070
Staphylococcus aureus 2 0.017 36
Therefore, the low yield of allicin in ATB cultures is presumed to result from incomplete substrate degradation.
Quite unexpectedly, Pseudomonas aeruginosa 2 exhib ited alliinase activity in this study, a contrast to previous findings. The positive result was given in triplicate tests, and the purity and identity of the cul ture were confirmed. Therefore, the change from alliinase- negative to alliinase-positive was presumed to be due to an alteration of the culture's characteristics, which occurred as a result of transfers or storage.
Inhibition Tests
Certain organisms cleave the powerful antibiotic, allicin, from its parent compound. This phenomenon was expected to lend a selective action to media containing alliin. Alliinase-positive organisms, inoculated onto solid media, might produce an inhibitory concentration of allicin. Such a localized production of allicin could arrest growth of the organisms while the cultures are still in the microcolony stage. Test organisms were streaked on STA in order to investigate the selectivity of alliin in media. The streaked plates were observed for colony formation after 24 and 48 h of incubation. The results of this test are recorded in Table 3. The occur rences of alliinase in the test organisms are listed for comparison. The anticipated results, growth of alliinase- negative cultures and inhibition of alliinase-positive cultures were not observed in this experiment. Although 37
Table 3
Growth of Selected Organisms on STA
Growth on STA Organism Alliinase 24 h 48 h
Pseudomonas aeruginosa 1 - + +
Flavobacterium rigense -- -
Serratia marcescens + + +
Enterobacter cloacae + + +
Escherichia coli 1 + + +
Escherichia coli 2 + + +
Escherichia coli 3 + + +
Bacillus megaterium 1 -- -
Bacillus megaterium 2 -- -
Bacillus circulans 1 - - -
Bacillus circulans 2 - - -
Bacillus cereus + - -
Bacillus firmus - - -
Bacillus brevis NC - -
Bacillus subtilis 1 + - -
Bacillus subtilis 2 + - -
Bacillus sphaericus - - -
Micrococcus luteus - + +
Staphylococcus aureus 2 - - +
Symbols: +, positive; negative; NC, not consistent in multiple tests. 38 some of the bacteria followed this pattern, there were also alliinase-negative strains which did not grow and alliinase-positive strains which did grow.
The concentration of alliin to be used in the STA medium had been arbitrarily chosen as 0.5%. Alliin it self, at this level was expected to not inhibit growth.
Yet, enzyme action should produce an ample amount of allicin to cause growth stasis. Varying the alliin con centration in the medium could produce differences in selectivity. Also, more sophisticated means of detecting inhibition might generate data of more significance.
However, the inhibition tests performed in this study did not indicate any correlation between alliinase activity and the ability to grow on STA, and this line of study was not pursued further.
Enzyme Induction
The use of glucose-salts media by previous workers, in studies of bacterial alliinases, suggests that alliinase production may be enhanced by growth in glucose- salts media. This hypothesis was tested by harvesting cells grown in NB and in GS, and assaying the cell crops for alliinase activity. Results of these tests are given in Table 4. Cell crops of alliinase-negative organisms, grown in NB, produced negative tests and, with exception of the Pseudomonas aeruginosa strains, would not culture in GS. Cell crops of alliinase-positive organisms con tained alliinase when harvested from GS. However, these 39
Table 4
Alliinase in Cell Crops of Selected Organisms
NB-grown GS-grown Organism cells cells
Escherichia coli 1 - +
Escherichia coli 2 - +
Enterobacter cloacae - +
Pseudomonas aeruginosa 1 - -
Bacillus subtilis - +
Bacillus brevis - 0
Staphylococcus aureus 1 - 0
Staphylococcus epidermidis - 0
Micrococcus luteus - 0
Symbols: +, positive; negative? 0, no growth in GS. 40 same alliinase-positive organisms had no detectable alliinase activity when they were cultivated in NB. The two pseudomonads were not found to contain alliinase, whether the cells were harvested from NB or GS. Cells of
Escherichia coli 1, cultured in ATB, were collected and tested for alliinase. The culture fluid gave a positive
NEM test, indicating that alliinase activity had occurred.
However, alliinase was not detected in the cell crop.
Inhibition or saturation of the enzyme might cause this lack of activity, but the phenomenon could also be ex plained by a low level of enzyme production. A small amount of enzyme might, during the 48 h incubation of the culture in ATB, bring about degradation of the alliin present. However, this quantity of enzyme may not be sufficient to produce detectable allicin during the 1 h incubation used for testing cell crops.
The enzyme induction experiments support a hypothesis that the alliinase system may be involved in allowing organisms to grow in GS. There is probably only a very small amount of alliinase synthesized in response to the alliin contained in ATB, and little or no alliinase pro duced in NB. However, if alliinase is involved in growth in minimal media, cells in GS would require a substantially increased level. The occurrence of detectable amounts of alliinase in GS-grown cells demonstrates a much greater enzyme activity than that found in NB-grown cells and ATB- grown cells. This suggests that alliinase may be a part 41
of the inducible enzyme makeup which is necessary for
growth in GS.
Alliinase Identity
The alliin to allicin cleavage plays no known role in
the metabolism of bacteria which are growing in glucose-
salts media. However, the present study shows that
alliinase activity is induced by culturing certain organ
isms in GS. It is logical to assume, then, that the alliinase reaction is not carried out by an enzyme which is specific for alliin cleavage. Rather, it is likely that the responsible enzyme is part of a major pathway and, only incidentally, degrades alliin. Considering the specificity of enzymes, alliin is probably structurally related to the natural substrate of this enzyme.
A number of workers have investigated the metabolism of amino acids by bacteria. Enzymes are known which degrade serine, threonine, tryptophan, cysteine, and cystathionine via a,3-elimination reactions (49). These were considered as possible sources of alliinase activity.
Of these enzymes and their reactions, alliinase activity most closely resembles the cleavage of cystathionine by
3-cystathionase. The mechanisms of both the alliinase and the 3-cystathionase reactions involve pyridoxal enzymes, which accomplish a,3-eliminations to form ammonia, pyru vate, and sulfur-containing fragments. Cystathionase in the liver of mammals decomposes several analogs of alliin by a similar mechanism, and is probably responsible for 42
the alliinase activity observed in mammalian systems.
Furthermore, a 3-cystathionase is found in the methionine
pathway of bacteria. This enzyme is involved in the
assimilation and metabolism of sulfur compounds, and would
probably be heavily used during growth in minimal media.
Extensive enzyme activity would be necessary in minimal
media, to supply the required sulfur compounds. Converse
ly, complex media contain many of these essential sulfur
compounds, and total synthesis would not be necessary.
This line of thought may explain why cell crops of
alliinase-positive organisms contain alliinase when grown
in GS, but do not contain detectable alliinase activity when grown in NB or ATB. Economy of energy and materials
would preclude excessive synthesis of the enzyme in the
complex media. Detectable levels of alliinase would occur
only in cells harvested from minimal media, where a greater
amount of the enzyme is needed for growth.
Cell crops of Escherichia coli 1, cultivated in GS,
possessed an induced alliinase. Efforts were made to
demonstrate that these cell crops were also adapted to
cystathionine degradation. Using pyruvate formation as an
index of enzyme action, both alliinase and cystathionase
activities were shown to be present in the GS-grown cells.
Chromatographic conformation of pyruvate was accomplished by comparison of values to a pyruvate standard (R^ =
0.72). No other solute spots were observed in the chro matograms . 43
Table 5 lists the quantitative results of this exper
iment. The values represent an average of duplicate tests.
Very little degradation of either alliin or cystathionine
was found in the NB-grown cells. These observations led
to the conclusion that both alliinase and cystathionase
activities are induced by growth of Escherichia coli 1 in
GS. Cells of Bacillus subtilis and Pseudomonas aeruginosa
1 were also cultivated in GS and tested for cleavage of pyruvate from cystathionine. The results obtained with
these two organisms were consistent with those predicted by the alliinase test. Bacillus subtilis cells possessed both alliinase and cystathionase activities when cultured
in GS. Cells of the pseudomonad degraded neither alliin nor cystathionine.
Based on the evidence available, it was assumed that alliin degradation in ATB cultures and GS-grown cells could be ascribed to an inducible cystathionase system.
In an effort to prove this assumption, an experiment was undertaken to demonstrate co-purification of alliinase and cystathionase activities from GS-grown cells of Escherichia coli 1. The results of this study are presented in Table
6. Approximately a sixty-fold purification of the enzyme was achieved in this experiment. The ratios of specific activities remain approximately the same through the puri fication procedure, indicating that alliinase and cysta thionase activities may be carried out by the same enzyme system. 44
Table 5
Pyruvate Production by Cells of Escherichia coli 1
Cell crop Substrate Pyruvate (mg)a/b
NB-grown 5 mg alliin 0.2
GS-grown 5 mg alliin 1.0
NB-grown 5 mg cystathionine 0.0
GS-grown 5 mg cystathionine 0.9
Values represent the average of duplicate tests. u Reagent blanks incorporated appropriate cell crops, to negate added pyruvate. Table 6
Purification of Alliinase and Cystathionase Activities
Specific activities (ymole/min/yg) Ratio of specific activities Purification step (cystathionase/alliinase) Alliinase Cystathionase
Crude extract 0.004 0.004 1.0
Protamine sulfate 0.011 0.014 1.3
First ammonium sulfate 0.114 0.148 1.3
Calcium phosphate gel 0.227 0.227 1.0
Second ammonium sulfate 0.245 0.262 1.1 46
Summary
A relatively simple procedure has been developed for detecting the occurrence of alliinase in bacteria.
Alliinase was found in some bacterial isolates, but not in others. In still other strains, alliinase activity was not consistent between tests. Alliinase-positive organ isms were found to produce the enzyme when grown in GS, but not when grown in NB. This fact led to the hypothesis that alliin cleavage might result from the action of a cystathionase. In bacteria, cystathionase activity is found as an intermediate step in the methionine pathway, a rather ubiquitous enzyme system which is involved in sulfate assimilation. It should be expected that this pathway, including cystathionase, would be expressed in cells cultivated in minimal media. Both alliinase and cystathionase activities were demonstrated in GS-grown cells of Escherichia coli 1. These activities were found to co-purify through a series of manipulations. Therefore, the hypothesis was supported, that alliinase activity is probably achieved via a cystathionase system. LITERATURE CITED
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Structures of Alliin and Related Compounds Discussed in the Text
Compound Structure
N-acetyl-(1,2-dichlorovinyl)-L-cysteine HOOC-CH(HN-CO-CHj) -CH2-S-CC1=CHC1 alliin see S-allyl-L-cysteine sulfoxide allocystathionine HOOC-CHNH 2-CH2-S-C H 2-CHNH 2-COOH
S-allyl-L-cysteine HOOC-CHNH 2-CH 2-CH 2CH=CH 2
S-allyl-L-cysteine sulfoxide h o o c -c h n h 2-c h 2-s o -c h 2-c h =c h 2
S-n-amylcysteine h o o c -c h n h 2-c h 2-s -c h 2-c h 2c h 2-c h 2-c h 3
S-n-butylcysteine HOOC-CHNH 2-CH 2-S-C H 2-CH 2-CH 2-CH 3
cystathionine HOOC-CHNH2-CH2-S-C H 2CH2-CHNH2COOH
L-cysteine HOOC-CHNH 2-CH 2-SH
cysteine methyl ester c h 3o o c -c h n h 2-c h 2-s h
cystine HOOC-CHNH 2CH 2-S-S-CH 2-CHNH 2COOH
desoxyalliin see S-allyl-L-cysteine
S- (1,2-dichlorovinyl)-L-cysteine HOOC-CHNH2-CH2-S-CCl=CHCl Table 1A (continued)
Compound Structure
S-(1,2-dichlorovinyl)-L-cysteine sulfoxide HOOC-CHNH 2-CH2-SO-CC1=CHC1 dihydroalliin see S-n-propyl-L-cysteine sulfoxide djenkolic acid HOOC-CHNH 2-CH 2-S-CH 2-S-CH 2-S-CH 2-CHNH 2COOH
S-ethyl-L-cysteine HOOC-CHNH2-CH 2-S-CH 2-CH 3
S-ethyl-L-cysteine sulfoxide h o o c -c h n h 2-c h 2-s o -c h 2-CH3 homocysteine HOOC-CHNH2-CH2-CH2-SH
S-isoamyl-L-cysteine h o o c -c h n h 2-c h 2-s -c h 2-c h 2-c h (c h 3)-c h 3
S-isobutylcysteine h o o c -c h n h 2-c h 2-s -c h 2-c h (c h 3)-c h 3 isovalthine h o o c -c h n h 2-c h 2-s -o c -c (c h 3)2-c o o h lanthionine h o o c -c h n h 2-c h 2-s -c h n h 2-c o o h y-methiobutyric acid h o o c -c h 2-c h 2-c h 2-s -c h 3 methionine h o o c -c h n h 2-c h 2-c h 2-s -c h 3
S-methylcysteine HOOC-CHNH2-CH 2-S-CH 3
S-methylcysteine sulfoxide HOOC-CHNH 2-CH 2-SO-CH 3 Table 1A (continued)
Compound Structure a-oxy-y-methiobutyric acid HOOC-CO-CH 2-CH 2-S-CH 3
S-propyl-enyl-L-cysteine sulfoxide HOOC-CHNH 2-CH 2-SO-CH=CH-CH 3
S-n-propylcysteine h o o c -c h n h 2-c h 2-s -c h 2c h 2-c h 3
S-n-propylcysteine sulfoxide h o o c -c h n h 2-c h 2-s o -c h 2-c h 2-c h 3 VITA
Lawrence A. "Larry" Reily was born May 27, 1949, in
Jackson, Mississippi. Since the age of one year, he has been a resident of Baton Rouge, Louisiana. He was grad uated from Istrouma High School in 1967 and began his college career at Louisiana State University. There, he received the degree of Bachelor of Science in 1971, in the Department of Microbiology. After graduation, he worked for Hart-Delta, Incorporated, where he was respon sible for quality control on the company’s products, veterinary pharmaceuticals. After one year, he left the company to continue his education at Southeastern Louisiana
University. There, he received the Master of Science degree in 1975, in the Department of Biological Sciences.
He was employed by Joan of Arc Company, a food canning concern, where he worked for two years, in quality assur ance. He then resumed his educational pursuits, at
Louisiana State University. At present, he is a candidate for the degree of Doctor of Philosophy in the Department of Food Science.
57 EXAMINATION AND THESIS REPORT
Candidate: Lawrence A. Reily
Major Field: Pood Science
Title of Thesis: Hie Occurrence of Allinase in Selected Bacterial Strains
Approved:
Major Professor and Chairman
v Dean of the Graduate C Scjsool
EXAMINING COMMITTEE:
— A ■
3 - 4^2
Date of Examination:
April 16,1980